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Purified Mouse Anti-Human PI3-Kinase C2β
Purified Mouse Anti-Human PI3-Kinase C2β

Western blot analysis of PI3-Kinase C2β on a HeLa lysate. Lane 1: 1:1000, lane 2: 1:2000, lane 3: 1:4000 dilution of the PI3-Kinase C2β antibody.

Purified Mouse Anti-Human PI3-Kinase C2β

Immunofluorescent staining of A549 (ATCC CCL-185) cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the alcohol perm protocol and the anti-PI3-Kinase C2β antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway 855 Bioimager using a 20x objective.  This antibody also stained U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells using both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Western blot analysis of PI3-Kinase C2β on a HeLa lysate. Lane 1: 1:1000, lane 2: 1:2000, lane 3: 1:4000 dilution of the PI3-Kinase C2β antibody.

Immunofluorescent staining of A549 (ATCC CCL-185) cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the alcohol perm protocol and the anti-PI3-Kinase C2β antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway 855 Bioimager using a 20x objective.  This antibody also stained U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells using both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing)
Mouse IgG1
Human PI3-Kinase C2β aa. 16-209
Western blot (Routinely Tested), Bioimaging (Tested During Development)
165 kDa
250 µg/ml
AB_398865
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Bioimaging

1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.

2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well.  Incubate for 10 minutes at room temperature (RT).

3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:

a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

     OR

b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.

5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.

6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.

7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.

8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.

9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.

10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

11. View and analyze the cells on an appropriate imaging instrument.

Bioimaging:  For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp

Western blot:  For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Triton is a trademark of the Dow Chemical Company.
611342 Rev. 3
Antibody Details
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22/PI3-K

Phosphatidylinositol (PtdIns) (3) kinase phosphorylates the D-3 position of the inositolring of PtdIns, producing PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3. PI3-kinase is a heterodimer of an 85 kDa regulatory subunit (p85) and a 110 kDa catalytic subunit (p110). However, it is only one member of a larger family of proteins with similarity to the p110 subunit. These different PI3-kinase isoforms have been divided into three classes. Class I consists of p110α and p110ß which bind the p85 subunit and associate with receptor tyrosine kinases. Class II includes 68D and cpk from Drosophila, p170 and cpk-m from mouse, and C2α, C2ß (HsC2), and C2γ from human. These proteins phosphorylate PtdIns and PtdIns(4)P, but not PtdIns(4,5)P2, and each contain a C-terminal C2 domain that may negatively regulate the catalytic domain. Class III members only phosphorylate PtdIns to PtdIns(3)P and include the S. cerevisiae Vps34p and its human homologs. In humans, the class II PI3-kinases C2α and C2ß have similar catalytic, PI kinase, and C2 domains. However they differ in their N-terminal regions. In addition, C2ß has no cation specificity, while C2α prefers Mg2+-ATP for optimal phosphorylation.

611342 Rev. 3
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
611342 Rev.3
Citations & References
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Development References (1)

  1. Arcaro A, Volinia S, Zvelebil MJ, et al. Human phosphoinositide 3-kinase C2beta, the role of calcium and the C2 domain in enzyme activity. J Biol Chem. 1998; 273(49):33082-33090. (Biology). View Reference
611342 Rev. 3

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.